Folate is a B vitamin acquired by dietary consumption of leafy green vegetables, whole grains and other food sources. Folic acid is the synthetic form of folate that may be taken in supplement form or as an additive to milled grain products and other manufactured/processed foods.
Folate is a B vitamin that is centrally involved in one carbon metabolism. It is important for facilitating cellular methylation reactions involving substrates such as DNA, proteins and lipids, as well as xenobiotics and prescription medications, and for generating thymidylate and purines (Stover, 2004, Nutr Rev 62:S3-12). RBC folate concentrations are generally measured as “total folate” without distinguishing between the several forms of folate that are present. This potentially limits the predictive value of such measurements. The major circulating form of folate is 5-methyltetrahydrofolate (5-CH3-THF or 5-MTHF). Intracellular 5-CH3-THF, derived from 5,10-methylenetetrahydrofolate, is used to remethylate homocysteine (Hcy) to methionine, which in turn is converted to the universal methyl donor S-adenosyl methionine. In the latter reaction, 5-CH3-THF is converted to tetrahydrofolate (THF). Alternatively, to facilitate nucleic acid synthesis, 5,10-methylenetetrahydrofolate is converted via 5,10-methenyltetrahydrofolate (5,10-methenylTHF) and formylated derivatives to THF. Thus, 5-CH3-THF, THF and 5,10-methenylTHF represent distinct folate derivatives that play key roles within the methylation and nucleic acid synthesis compartments of folate/Hcy metabolism.
Folate/Hcy metabolism provides one-carbon units for many essential biological processes (Selhub, 2002, J Nutr Health Aging 6(1):39-42; Stover, 2004, Nutr Rev 62:83-12; Smulders et al., 2005, Semin, Vase. Med, 5(2):87-97; Huang et al., 2007, Biomed, Chromatogr. 21:107-12). In particular, it enables the methylation of substrates including DNA, proteins and lipids, and the generation of thymidylate and purines, important functions that require different intracellular folate derivatives (Brown et al., 2006, Atherosclerosis 189:133-41). Low folate status is associated with elevated levels of circulating homocysteine (hyperhomocysteinemia), (Huang et al., 2007, Biomed. Chromatogr. 21:107-12) and a phenotype characterized by low red blood cell (RBC) and serum folate together with high homocysteine has been implicated in many diverse human pathologies ranging from neural tube defects such as spina bifida (Pitkin, 2007, Am. J. Clin. Nutr. 85:285S-288S; Jensen et al., 2006, Am. J. Med. Genet. A 140:1114-8) to aging-related conditions such as cardiovascular disease (Smulders et al., 2005, Semin. Vase. Med. 5(2):87-97) and colorectal cancer (Kim, 2007, Mol. Nutr. Food Res 51(3):267-92; Sanderson et al., 2007, Br. J. Nutr. 98(6):1299-304). In addition, individuals with a low folate/high homocysteine phenotype are at elevated risk of many pathologies including, but not restricted to, coronary artery disease, cerebrovascular disease, peripheral vascular disease, thrombosis, inflammatory bowel disease, Alzheimer's disease, some cancers, and some neuropsychiatric diseases. In pregnancy, the phenotype has been associated with spina bifida, Down syndrome, early spontaneous abortion, premature birth and pre-eclampsia. In the elderly, the phenotype is associated with cognitive decline. Folate dysregulation negatively impacts several key cellular functions.
In addition, the potentially deleterious effects of hyperhomocysteinemia are a consequence of inadequate levels of the methyl donor 5-methyltetrahydrofolate (5-MTHF) (Brown et al., 2006, Atherosclerosis 189:133-41; Blount et al., 1997, Proc. Natl. Acad. Sci. USA 94(7):3290-5). Folate/homocysteine metabolism also modulates glutathione biosynthesis through the cystathionine/cysteine pathway, which is in turn crucial for controlling intracellular redox status (Zhu et al., 2008, Rapid Commun. Mass Spectrom. 22(4):432-40).
Mild hyperhomocysteinemia, characterized by high (>13 μmol/L) circulating concentrations (Jacques et al., 1999 N Engl J Med 340:1449-54) of the intermediate amino acid homocysteine, has been associated with a wide range of human pathologies including cardiovascular disease (Refsum et al., 1998 Ann Rev Medicine 49:31-62), stroke (Furie et al., 2006 Semin Neurol 26:24-32), Alzheimer disease (Seshadri et al., 2006 J Alzheimers Disease 9:393-8), cognitive impairment (Durga et al., 2007 Lancet 369:208-16), inflammatory bowel disease (Mahmud et al., 1999 Gut 45:389-94), some cancers (Powers et al., 2005 J Nutr 2005 135:2960S-66S), and adverse reproductive outcomes, including birth defects such as spina bifida (Mitchell et al., 2004 Lancet 364:1885-95). In a clinical setting, measurements of plasma total Hcy (tHcy) are conducted for a variety of purposes such as cardiovascular disease risk assessment and the management of patients taking anti-folate drugs. As elevated mild hyperhomocysteinemia is often underpinned by sub-optimal folate status (Jacques et al., 1996 Circulation 93:7-9; Harmon et al.,1996 Q J Med 1996 89:571-7), concurrent measurements of plasma folate and red blood cell (RBC) folate are often conducted.
There is a well-established reciprocal relationship between folate and tHcy (Jacques et al., 1996 Circulation 93:7-9; Harmon et al., 1996 Q J Med 89:571-7). In addition to hyperhomocysteinemia, low folate status can lead to impaired methylating capacity, compromised nucleic acid synthesis, and increased glutathione production, all of which have pathogenic potential (Stover et al., 2004 Nutr Res 62: 3-12). The importance of adequate folate status for preventing spina bifida is well established (Czeizel, 1992 N Engl J Med 327:1832-5; Barry et al., 1999 N Engl J Med 341:1485-90). Indeed, to reduce the prevalence of birth defects such as spina bifida, folic acid fortification of milled grain products was mandated in the United States in 1998.
Over the past two decades, many clinical studies have sought to characterize folate/Hcy metabolism in order to identify biochemical features that are associated with particular pathologies. In addition, studies of folate/Hcy phenotypes in healthy populations have been undertaken to examine the determinants of plasma and RBC folate levels and to assess the need for, or consequence of, folic acid supplementation and fortification. Many of these studies have relied on biochemical measurements that have been made in clinical laboratories using standard analytical methods and, for RBC folate, have considered only total folate levels.
Over the past ten years several functional polymorphisms in enzymes involved in folate/homocysteine metabolism have been described (Schwahn et al., 2001, Am. J. Pharmacogenomics 1(3):189-201). The functional polymorphism with the most readily observed impact on phenotype is the C to T transition at nucleotide 677 (677C>T) of the methylenetetrahydrofolate reductase (MTHFR) gene, which results in a change in amino acid residue from Ala>Val at position 222, located at the bottom of the (βα)8 barrel of the catalytic domain of the enzyme (Pejchal et al., 2006, Biochemistry 45(15):4808-18). The 677T allele encodes an enzyme that is ‘thermolabile’ and less efficient at generating the 5-MTHF that is needed for both homocysteine remethylation and the generation of S-adenosyl methionine for methylation reactions. It is well established that MTHFR 677TT homozygotes with low folate status are at greatly increased risk of being hyperhomocysteinemic (Strain et al., 2004, Proc. Nutr. Soc. 63(4):597-603). Selhub and colleagues have established that in the RBCs of individuals with this genotype, 5-MTHF comprises only 60% of total RBC folate, whereas this form predominates in the RBCs of their 677CC peers (Bagley et al., 1998, Proc. Natl. Acad. Sci USA 95(22):13217-20; Davis et at, 2005, J. Nutr. 135(5):1040-4). Subsequently, it has been shown that the MTHFR C677T genotype is the primary determinant of non-methylfolate accumulation in RBCs (Botta et at, 2000, Am. J. Epidemiol. 151(9):862-77). The homozygous MTHFR 677TT genotype confers a significantly increased risk of many of the conditions with which a low folate, high homocysteine phenotype has been associated, for example, approximately 2-fold for spina bifida (Lewis et at, 2005, BMJ. 331(7524):1053), and 1.15-fold for cardiovascular disease (Smulders et al., 2007, J. Nutt Biochem. 18(10):693-9). However, the excess individual risk of developing such conditions in relation to their prevalence is insufficient to warrant genetic testing and counseling. Therefore, there is a need to establish laboratory methods to define the degree of variation in the ‘folate phenotypes’ between and within the three MTHFR 677C>T genotypes in order to determine whether there are subsets of TT homozygotes, and possibly of CT heterozygotes, with extreme phenotypes that may be associated with greatly enhanced risk. Individuals falling into such subsets might benefit from early identification and intervention.
Several drugs have been designed to disrupt specific aspects of folate/homocysteine metabolism in order to produce therapeutic effects in the context of a wide range of disease including auto-immune/inflammatory disease and cancer. For example methotrexate (MTX) is widely used in rheumatoid arthritis (as well as leukemia) and 5-fluorouracil (5-FU) is the mainstay of many solid tumor treatment protocols.
Clinical laboratory tests are available to measure serum/plasma folate and Red Blood Cell (RBC) folate. The latter is currently the preferred mean for assessing recent folate history as RBCs retain the folates that are present at erythropoiesis through their 120-day life cycle. Such tests are used, sometimes in conjunction with Hcy measurements, to determine whether folic acid supplements should be prescribed/recommended to: correct deficiency/insufficiency; reduce the risk of cardiovascular disease, birth defects and other pathologies; mitigate the side effects of drugs with anti-folate properties, including MTX and 5-FU.
Cellular folate, including RBC folate, is not a single chemical entity. There are several forms of folate and each participates in different metabolic processes, including methylation and nucleic acid synthesis. The ability to measure “folate” using standard tests therefore provides only quantitative information and lacks the qualitative resolution to provide information regarding the support of diverse cellular functions, the disruption of which might be pathogenic, and require remediation.
In humans, defects of neurulation are relatively common and result in serious malformations, including anencephaly and spina bifida (Mitchell et al., 2004, Lancet 364(9448):1885-1895). Collectively, these malformations are referred to as neural tube defects (NTDs). While a small proportion of fetuses/infants affected by an NTD are identified as having an underlying syndrome, no specific cause(s) can be identified in the majority. However, a portion of NTDs in this latter group can be prevented by maternal periconceptional supplementation with folic acid (1991 Lancet 338(8760):131-137; Czeizel et al., 1992, N Engl J Med 327(26):1832-1835). In the absence of such supplementation, pregnancies that result in NTD birth outcome are characterized by low maternal folate status (Kirke et al., 1993, Q J Med 86(11):703-708; Mills et al., 1995, Lancet 345(8943):149-151). This suggests that folic acid is corrective rather than pharmacologically active, and therefore that there may be a causative relationship between maternal folate insufficiency (or dysregulation) and failure of neurulation very early in development. However, the precise mechanism(s) by which low folate status contributes to NTD etiology remains unknown.
The protective effect of maternal periconceptional folic acid has generated considerable interest in the identification of genetic variants that are associated with the risk of NTDs due to their impact on folate transport, metabolism or excretion (Beaudin et al., 2007, Birth Defects Res C Embryo Today 81(3):183-203). However, only one such variant, the 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C>T single nucleotide polymorphism (SNP), has been strongly (although not unequivocally) implicated as an NTD risk factor (Barber et al., 2000, Mol Genet Metab 70(1):45-52; Shields et al., 1999, Am J Hum Genet 64(4):1045-1055). Consequently, there is interest in expanding the list of genetic candidates for NTDs to include genes that are biologically linked to other known or suspected NTD risk factors, including maternal obesity, diabetes, and hyperthermia (e.g. fever).
Many of the known NTD risk factors (e.g. diabetes, obesity) have inflammatory features, suggesting that genes involved in the inflammatory response may be involved in the etiology of this group of conditions. Interestingly, it has been shown, in a cultured endothelial cell line, that folate insufficiency induces increased synthesis and export of monocyte chemoattractant protein 1 (MCP-1), a potent pro-inflammatory chemokine (Brown et al., 2006, Atherosclerosis 189(1):133-141). In turn, MCP-1 acts in an autocrine fashion to elicit changes in cell morphology, including the acquisition of actin stress fibers (Brown et al., 2006, Atherosclerosis 189(1):133-141). Since actin dynamics underpin cellular shape changes such as those required for convergent extension during neurulation (Schoenwolf et al., 1990, Development 109(2):243-270), alterations of MCP-1 levels could have a direct effect on morphogenesis. Furthermore, MCP-1, together with other chemokines and cytokines, appears to be important in signaling between the embryo and endometrium during implantation and placentation (Kayisli et al., 2002, Am J Reprod Immunol 47(4):213-221), which occurs just prior to neurulation. Hence, altered MCP-1 expression could also influence the risk of NTDs through modulation of maternal inflammatory responses.
The (-2518) A>G promoter polymorphism of CCL-2, the gene encoding MCP-1, confers differential responsiveness to Interleukin-1 (IL-1) (Rovin et al., 1999, Biochem Biophys Res Commun 259(2):344-348)and is, therefore, a logical NTD genetic candidate that is only indirectly related to folate metabolism. Evaluation of this polymorphism in a large number of spina bifida ease-parent triads indicated that maternal CCL-2 genotype is associated with the risk of spina bifida in offspring (Jensen et al., 2006, Am J Med Genet A 140(10):1114-1118). Specifically, women with the CCL-2 (-2518) AA genotype appear to be at higher risk of having offspring affected with spina bifida than women with the AG or GG genotypes. As monocytes from CCL-2 AA homozygotes are known to produce less MCP-1 (as compared to those from CCL-2 AG heterozygotes or GG homozygotes) in response to IL-1, the observed increased risk of spina bifida in the offspring of women with the CCL-2 (-2518) AA genotype was hypothesized to be due to a sub-optimal systemic and/or local immune or inflammatory response resulting from low MCP-1 levels at the time of neural tube closure. However, as the CCL-2 (-2518)A>G polymorphism is not the only determinant of MCP-1 levels this explanation may be overly simplistic.
In addition to the CCL-2 (-2518)A>G polymorphism, variables that have been associated with MCP-1 levels include sex, age, race, diabetes, obesity, smoking status and the region of chromosome 3 that contains the chemokine receptor gene cluster, which includes the receptor for MCP-1 (Bielinski et al., 2007, Genes Immun 8(8):684-690; McDermott et al., 2005, Circulation 112(8):1113-1120). However, there are no published studies that have focused on the potential determinants of MCP-1 levels in reproductive age females.
There exists a long-felt need to develop novel methods to accurately identify and quantify the key forms of folate which are present in different parts of folate/homocystein metabolism and that have the potential to differentially diagnose and assess the risk of particular pathologies, and to predict/monitor responses to vitamin supplements (folic acid and other B vitamins) and anti-folate medications. In addition, there is a need to develop novel methods to accurately identify genetic and environmental variables that influence MCP-1 levels in women at risk of having an NTD affected pregnancy. The present invention meets these needs.